Sahil Parikh- Associate Director, Strategic Marketing, Charles River Microbial Solutions; Jim Polarine Jr., MA.- Senior Technical Service Manager, STERIS Corporation; Stacey Ramsey, MSc- Senior Technical Services Manager, Charles River Microbial Solutions; Yong Jian Lee, PhD- Technical Services Manager, Charles River Microbial Solutions
While on the leading edge of many medical breakthroughs, cell and gene therapy manufacturers often face historically problematic concerns when controlling for contamination in their cleanroom facilities. Even though advancements are being made in the equipment and technique, the final therapeutic products cannot be terminally sterilized and much of the manufacturing process remains manual and aseptic, which presents a high risk for contamination. Moreover, as traditional pharmaceutical and biopharma manufacturers are aware, the prevention, detection, and remediation of fungal environmental contaminants are an ever-present cause for concern. Due to the inherent nature of production and administration of medicines, such as short shelf-life and advanced therapy medicinal products, there is a higher risk to patient safety than ever before.
Rapid microbiological methods provide advantages in helping to control fungal contamination; however, they have historically faced several challenges when applied to cell-based therapies. Studies on the performance of a novel rapid microbiological detection system with a specific focus on the ability to detect mold isolates that frequently occur within a cell and gene therapy manufacturing environment have been performed. The optimization of growth conditions for detecting these high-risk isolates to ensure recovery of potential contaminants and subsequent rapid detection have also been performed.
The inability to control fungal contamination in cell and gene therapy facilities is largely due to failures in the prevention of ingress through equipment pass-throughs and personnel and from improper disinfection of surfaces and equipment inside the manufacturing facilities. Fungal spores, specifically, rest in the middle of the spectrum of possible contaminants in cleanrooms and cold rooms with a range of disinfectant susceptibility, as shown in Figure 1, but also above many bacteria that are typically controlled for via thorough sanitization. Therefore, facility cleaning procedures solely developed to control for vegetative bacteria are inadequate. They also need to demonstrate efficacy against fungicidal and sporicidal agents.
A risk assessment approach should be taken to prevent, monitor, and control bacterial and fungal contaminants. Recent United States Food and Drug Administration (FDA) 483 warning letters have revealed insufficient disinfection procedures to prevent fungal contamination of drug products purporting to be sterile, one of which was as recent as March 2022. Furthermore, many of these citations involve the improper validation of such methods.
A deeper root cause analysis has found that many contamination control strategies among cell and gene therapy facilities rely on using 70% alcohol alone. A hydrogen peroxide/peracetic acid blend used alone or in combination with 70% alcohol, following appropriate contact time is more effective, as shown in Figure 2.
Contamination Control Strategies Using Disinfectants
Commonly isolated cleanroom fungal isolates also tend to have their own resistance scale, with Chaetomium and Aspergillus species being among the most resistant, as shown in Figure 3. These two mold genera have been frequently cited as the source of outbreaks due to a lack of adequate control and improper use of sporicides. They can often reside in hard-to-reach or hard-to-disinfect areas, such as drywall, door hardware, and material carts. Mold can produce significant problems within any drug production facility. Moreover, the overuse of sporicides and improper use are often cited, indicating a failure to address the underlying cause of the mold contamination and to control the resulting problem.
Developing and adopting a long-term strategy to control fungal spores and prevent outbreaks effectively begins with using appropriate sporicides at specific intervals within a validated cleaning and disinfection program. An in-situ evaluation is a mechanism to evaluate the overall effectiveness of a disinfection program to determine effectiveness under worst-case contamination conditions such as a facility shutdown or crisis event. An evaluation of an effective in-situ validation was performed in 2021, at a pharmaceutical sterile manufacturing facility utilizing a triple-cleaning framework involving a low pH phenolic disinfectant, high pH phenolic disinfectant, then followed by a hydrogen peroxide/peracetic acid sporicide. This type of cleaning strategy helps to prevent the growth of both mold spores and bacterial spores. The study demonstrated the efficacy of such a program in contrast to in-vitro studies, representing real-world, worst-case scenarios such as when installing new equipment, room pressure modification, and new facility construction and in high traffic/high contact areas. Environmental monitoring was performed as a baseline to demonstrate the efficacy of a triple-cleaning procedure. Samples were taken before and after serial cleaning, steps using the low pH phenolic, the high pH phenolic, and then a ready-to-use blend of hydrogen peroxide/peracetic acid. The wet contact time for each disinfectant and sporicide was 10 minutes, with no precleaning or rinsing conducted between the steps. Additional details of the method for evaluation can be found in the published American Pharmaceutical Review article In-Situ Disinfectant Validation Case Study. 18
The outcomes of this study, which evaluated disinfection in ISO-5 and ISO-7 classified areas, found progressive and significant reductions of microbial counts after the final hydrogen peroxide/peracetic acid sporicidal agent was used. A risk assessment determined that the use of material carts within the material transfer area, in conjunction with high personnel activity, was the likely root cause. As described in the case study, the validated procedure demonstrates the efficacy and appropriate development of a cleaning and disinfection program, such as triple-cleaning. Furthermore, industry regulatory authorities, such as United States Food and Drug Administration (FDA), Health Products Regulatory Authority (HPRA), Brazilian Health Regulatory Agency (ANVISA), National Administration of Drugs, Foods and Medical Devices (ANMAT), National Agency for the Safety of Medicines and Health Products (ANSM), China Food and Drug Administration (CFDA), and Medicines and Healthcare Products Regulatory Agency (MHRA) view the in-situ validation data gathered during the triple cleaning have significant value in the disinfectant validation process.
Traditional pharmaceutical and biopharma manufacturing facilities have matured processes, personnel, and equipment, and have likely encountered or performed investigations and remediations for previous contamination events within their facilities. One should not overlook the collective knowledge and experience in this industry, which has not yet been developed in the emerging field of cell and gene therapies. While contamination prevention and routine disinfection are paramount and form the foundation of a risk-based approach to environmental monitoring for fungal contaminants, there is still an ever-present risk of an outbreak which will ultimately lead to contaminating the product manufactured. Therefore, in conjunction with validated disinfection and holistic prevention, rapid microbiological methods should be employed to provide a faster means to detect potential fungal contaminants in the cell or gene therapy product, while also reliably detecting a range of microorganisms.
Contamination Control Strategies Using Rapid Microbiological Methods
A framework for preventing and detecting possible contaminations is paramount for the cell and gene therapy industry. Many regulatory bodies offer updated guidance specific to the industry, such as the upcoming United States Pharmacopeia Chapter <1114>Microbial Control Strategies for Cell Therapy Products. Incorporating an environmental control strategy must also be combined with a quality testing strategy that can detect microbial contamination sooner and offer a robust detection mechanism that can be widely applied to all kinds of microorganisms, especially molds. An assessment of high, moderate, and low-risk areas or aseptic process points where contamination could be introduced should be monitored, with the microbial testing strategy modulated based on risk. This assessment includes both starting materials, such as apheresis, blood, or cells, to final product sterility. Rapid and alternative detection methodologies have been encouraged, especially those that can provide a faster, objective result that may be more sensitive to the traditional test. However, this poses a challenge to both suppliers and end-users. The detection of microorganisms using Adenosine trisphosphate (ATP) Bioluminescence has been widely considered a gold standard rapid microbial detection method since its discovery in the late 1970’s, because all microorganisms produce ATP. Several rapid microbiological detection technologies are available for the industry utilizing this detection technology; however, the presence of therapeutic cells represents a known limitation since the sample itself contains eukaryotic cells producing ATP.
The Celsis Adapt™ assay used in this study incorporates a cell lysis step, filtration, and concentration procedure for removal of eukaryotic cells, as well as an apyrase reagent for reducing free ATP, overcoming these known limitations of using ATP bioluminescence for microbial detection within cell therapies. After this treatment, a standard ATP bioluminescence assay can discern microbial contaminants in the cell therapy sample. The cell walls of these therapeutic cells differ in composition from microbial cells and ATP bioluminescence can therefore be differentiated from prokaryotic cells by applying different osmotic pressures during a lysis pre-treatment. Thus, the Celsis Adapt assay can provide quality control laboratories with an effective tool to monitor the contamination of final products and various intermediary products along manufacturing process.
However, evaluating the microbial detection range for any alternative or rapid microbiological method should include both slow-growing bacterial species and fungal species. As referenced above, the fungal genera Aspergillus and Chaetomium have been both difficult to control for and have posed a challenge in the consistent detection within a specified test period. Rapid detection methods, such as respiration-based detection, have reported varying range in the published time-to-detection results for molds. These range from of approximately two days to five days to no detection, even with method modification or additional media.
Mold Detection Performance Results
The following study demonstrates mold detection performance in several species within a specified time. A review of the detection range for Celsis Adapt shows broad applicability towards microbial contaminants within the cell therapy space, including a recent inoculation study with Chimeric Antigen Receptor cells (CAR T-cells).
This study was commissioned to review the applicability of the Celsis Adapt method towards a variety of fungal contaminants at inocula levels under 50 CFU. Species targeted for the study were recent recalls and known contaminants of particular interest in cell manufacturing facilities. Fungal inocula were added to TSB media and incubated at 20-25°C in triplicate to mimic compendial growth conditions. The results of the study can be found in Figure 4.
These studies found that fungal contaminants can be rapidly and confidently detected using Celsis Adapt with a specified period, in addition to the vast array of other microbial contaminations. Results determined using a high (5000 RLU) cutoff find microbial contamination for all replicates by five days using Celsis Adapt.
Conclusion
Fungal spores continue to be a contamination issue in the biopharma and cell and gene therapy industries. Having a robust contamination control strategy is essential to controlling the levels of bioburden in the cleanroom. Pass thru decon needs to be a consistent area of focus as fungal spores are routinely isolated on items entering the cleanroom such as carts, cartwheels, bags, intervention tools, equipment, magic markers, and cellphones. Biosafety hood cleaning and disinfection also require routine application of a sporicide to address the risk of fungal spores. A robust contamination control strategy in cell and gene therapy cleanrooms has an effective rotation program with a disinfectant and a sporicide. The Celsis Adapt is a very valuable method for detection of bioburden including fungal spores in cleanroom operations. This rapid system is very useful in CAPA (Corrective and Preventative Action) investigations for early detection of contaminants and relaying accurate and precise organism identification through accurate genotypic identification methods. Shortening the identification period for microorganisms in the cleanroom can shorten CAPA investigations and free up more time for production.
References
- Polarine, J., Bartnett, C., Klein, D., (2013) Fungal and Bacterial Spores: Contamination and Disinfection, 230.
- Mertens, A., Polarine, J., (2019) Contamination Control: Bringing Materials into the Cleanroom
- USP 43 <1072>Disinfectants and Antiseptics
- Draft 13 Annex 1 and MHRA Orange Guide (2021)
- Food and Drug Administration (FDA) (2004) FDA Guidance for Industry, Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice. September.
- Industry Articles (Ex. Dr. Scott Sutton, Jose Martinez, Dr. Tim Sandle, Richard Prince, Rebecca Smith, Jeanne Moldenhauer, Crystal Booth)
- PDA Cleaning and Disinfection TR No. 70 (October 2015)
- PDA TR No. 69 on Biofilms (2015)
- The CDC Handbook - A Guide to Cleaning & Disinfecting Cleanrooms (Dr. Tim Sandle 2016)
- A Guide to Disinfectants and their use in the Pharmaceutical Industry (Pharmig 2018)
- USP 43 <1116>Microbiological Control and Monitoring of Aseptic Processing Environments
- USP 43 <1115>Bioburden Control of Non-Sterile Drug Substances and Products
- PIC/S Guide to Good Practices for the Preparation of Medicinal Products in Healthcare Establishments (2014)
- WHO Annex 6
- PHSS Technical Monograph #20 “Bio-contamination characterization, control, monitoring and deviation management in controlled/GMP classified areas
- IEST-RP-CC018.5 Cleanroom Housekeeping: Operating & Monitoring Procedures (2020)
- Polarine, Jim & Chai, Richard & Kochat, Harry & Pulliam, Paul & Zhi, Kaining & Brooks, Kiara. (2021). In-Situ Disinfectant Validation Case Study. 10.13140/RG.2.2.15088.99844.
- Polarine J., Klein, D., Testing Fungi Common to Cell and Gene Therapy Facilities. Cleanroom & Processes 1, Nr. 1, 44–50 (2022)
- McDonnell, G.E. “Antisepsis, Disinfection, and Sterilization: Types, Action, and Resistance, ASM Press, Washington, DC, 2007.
About the Authors
Sahil Parikh is the Associate Director of Strategic Marketing at Charles River Microbial Solutions. He has over 14 years’ experience in the pharmaceutical quality control industry, spending half of that time as a quality control microbiologist using, developing, and validating multiple rapid method technologies. Having transitioned from the QC laboratory to a commercial role, Sahil now supports the marketing, branding, and customer experience for Charles River’s QC testing portfolio. Sahil Parikh received his Bachelor of Science degree in Biology from the University of Connecticut-Storrs.
Mr. Polarine is a senior technical service manager at STERIS Corporation. He has been with STERIS Corporation for twenty two years. His current technical focus is microbial control in cleanrooms and other critical environments. Mr. Polarine is a 2019 PDA Michael S. Korczynski Award recipient. He has lectured in North America, Europe, Middle East, Asia, and Latin America on issues related to cleaning and disinfection, microbial control in cleanrooms and validation of disinfectants. Mr. Polarine is a frequent international industry speaker and published several PDA book chapters and articles related to cleaning and disinfection and contamination control. He is active on the PDA’s COVID-19 Task Force. He was a co-author on PDA’s Technical Report #70 on Cleaning and Disinfection and Technical Report #88 on Microbial Deviations. Mr. Polarine teaches industry regulators as well as the pharmaceutical, biotech, and medical device industries at the PDA and the University of Tennessee. Mr. Polarine currently teaches the cleaning and disinfection course as part of the PDA Aseptic Processing Course, IEST, and at the University of Tennessee Parenteral Medications Course. Mr. Polarine is the current President for the PDA Missouri Valley Chapter and Technical Coordinator for the IEST. He is also a leader on the PDA’s Chapter Council Steering Committee. Mr. Polarine graduated from the University of Illinois with a Master of Arts in Biology. He previously worked as a clinical research manager with the Department of Veterans Affairs in St. Louis, MO and as a biology and microbiology instructor at the University of Illinois. His main hobby is storm chasing and is very active in tornado research and tornado safety.
Stacey Ramsey is the Senior Manager of our Celsis Technical Services and Validations laboratory in North Carolina. She is a career microbiologist with over 15 years of experience in the pharmaceutical industry working on drug product and endotoxin testing, environmental monitoring, process and method development and validation, and implementing new process, including rapid microbiology. Stacey has a master’s degree in environmental studies from Friends University in Wichita, Kansas and a bachelor’s degree in biological sciences from Wichita State University
Dr.Lee Yong Jian is Technical Services Manager at Charles River Laboratories and oversees the performance of Celsis ATP bioluminescence studies Singapore. He has helped various customers demonstrate suitability of their pharmaceutical products, conducting trainings for customers, and is also involved in the product development and validation of new reagents and test methods. Yong Jian received his PhD degree in Environmental Microbiology and Bachelor’s degree in Life Sciences from the National University of Singapore.
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